Calcioum and Phosphate Homeostasis/Vitamin D

Question 1

Where is the body’s calcium stored?

Answer 1

• The vast majority (99%) of the body’s calcium is stored as hydroxyapatite crystals in the endoskeleton, thereby playing a crucial structural role.
• The remaining 1% of the body’s calcium serves other important functions.

Question 2

What does calcium do?

Answer 2

• Calcium is essential for many fundamental processes including fertilization, vision, muscle contraction, nerve conduction, blood clotting, exocytosis, cell division, activity of enzymes, and intracellular signaling.
• The concentration of calcium (as well as phosphate) in the cellular and extracellular fluids must be maintained within a relatively narrow range (total Ca: 8.5-10.5 mg/dL; P: 3.0-4.5 mg/dL).

Question 3

The complex system of controls for calcium and phosphate homeostasis involve […], […] and […].

Answer 3

The complex system of controls for calcium and phosphate homeostasis involve parathyroid hormone (PTH), calcitonin (CT) and vitamin D (active form = 1,25-dihydroxyvitamin D3).

•and estrogen!

Question 4

PTH and Calcium Homeostasis

Answer 4

• The acute modulation of calcium is mediated by PTH.
• A low plasma concentration of calcium stimulates the release of PTH by the parathyroid glands.
• PTH, in turn, causes the resorption of calcium from the pool in bone while also enhancing the reabsorption of calcium in the kidney.
• PTH produces an indirect effect on intestinal calcium absorption by increasing the conversion of vitamin D to its active form in the kidney

Question 5

Vitamin D and Calcium Homeostasis

Answer 5

• Long-term (chronic) adjustments in plasma calcium are accomplished by altering the absorption of dietary calcium, thus preventing a significant drain on the skeletal mineral.
• Vitamin D regulates intestinal calcium absorption by stimulating calcium uptake as well as by promoting renal reabsorption.

Question 6

Estrogen (and Testosterone) in Calcium Homeostasis

Answer 6

•The role of estrogen (and testosterone) in this scenario is the prevention of bone resorption.

Question 7

Phosphate Homeostasis

Answer 7

• In terms of phosphate homeostasis, vitamin D promotes intestinal absorption and renal reabsorption.
• By its concomitant effects on calcium and phosphate, vitamin D maintains the Ca•PO4 matrix, thereby favoring bone mineralization.
• The lack of vitamin D leads to bone loss – rickets in children and osteomalacia in adults.
• With the primary signal for the release of PTH being a low concentration of calcium, the marked mobilization of phosphate during resorption of bone, together with the increased intestinal absorption of phosphate due to vitamin D, could potentially lead to hyperphosphatemia.
• To offset this problem, PTH prevents hyperphosphatemia by diminishing phosphate renal reabsorption in the proximal tubule.
• Consequently when plasma PTH is elevated, phosphaturia can be observed.

Question 8

Answer 8

Question 9

Answer 9

Question 10

Answer 10

Question 11

Answer 11

Question 12

PTH Biosynthesis and Secretion

Answer 12

•PTH is initially biosynthesized as prepro-PTH. The biosynthetic and secretion pathway for PTH, in the chief cells of the parathyroid gland, resembles that of prepro-insulin translation and sequential cleavages to insulin. Processing leads to storage in granules that ultimately fuse with the cell membrane to release their contents. Unlike insulin, but like glucagon, PTH has a single polypeptide chain.

Question 13

The biosynthesis and secretion of PTH is regulated by […].

Answer 13

• The biosynthesis and secretion of PTH is regulated by plasma calcium.
• The calcium sensor is a Gq protein-coupled receptor that acts to “trap” serum calcium.
• Like all G-protein coupled receptors, the CaR has 7-transmembrane helices.
• Activated Gq, stimulates phospholipase C that cleaves phosphatidylinositol in the inner leaflet of the plasma membrane to promote the formation of the second messenger, inositol trisphosphate (IP3), as well as diacylglycerol (DAG).
• IP3 promotes release of calcium from intracellular stores in the endoplasmic reticulum and, together with the likely influx of extracellular calcium, inhibits secretion of PTH.
• This elevated intracellular calcium in some way blocks fusion with the plasma membrane of the PTH-containing storage granules.
• Additionally, elevated 1,25(OH)2D3 inhibits transcription of the PTH gene to decrease production of PTH.

-Since 1,25(OH)2D3 raises plasma calcium, its inhibitory effect on PTH prevents hypercalcemia.

•When the plasma concentration of calcium is low, this inhibitory mechanism is inactive.

Question 14

Biochemical Action of PTH in the Kidney

Answer 14

• PTH in the circulation binds to its receptor on cells in the target tissues (i.e., bone, kidney). The activated PTH receptor complexes with Gs protein and therefore is coupled to activation of adenylyl cyclase to increase cAMP.
• Because the kidney is a primary site of PTH action, PTH infusion sharply increases urinary excretion of cAMP in humans.
• This rise in cAMP precedes a marked augmentation of urinary phosphate that results from PTH inhibiting renal phosphate reabsorption.
• In the renal proximal tubule, phosphate is reabsorbed on a sodium-phosphate cotransporter that is driven by the sodium gradient.
• PTH reduces phosphate reabsorption by decreasing the number of transporters in the membrane.

Question 15

Biochemical Action of PTH - Bone

Answer 15

• PTH in the circulation binds to its receptor on cells in the target tissues (i.e., bone, kidney).
• The activated PTH receptor complexes with Gs protein and therefore is coupled to activation of adenylyl cyclase to increase cAMP.
• In bone, PTH acts via cAMP in osteoblastic cells.
• The osteoblasts, in response to PTH, increase resorptive (paracrine) factors like IL-6 that acutely stimulate osteoclastic resorption of bone mineral.
• PTH also induces, on the surface of osteoblasts, the expression of RANKL [receptor activator of NF-kB ligand]/ODF [osteoclast differentiating factor], which causes a net enhancement in the number of osteoclasts.
• The ligand binds to a receptor located on the surface of monocytic osteoclast progenitor cells (MOPs). RANKL/ODF, in combination with M-CSF (macrophage colony stimulating factor) that binds to the c-Fms receptor, causes the precursor cells to differentiate and fuse to form multinucleate osteoclasts.
• This effect increases the capacity for bone resorption.

Question 16

Calcitonin

Answer 16

• Calcitonin reverses the action of PTH on bone resorption to cause a fall in blood calcium.
• CT secretion by the C-cells of the thyroid is enhanced by high calcium in the blood.
• CT is physiologically important postprandially, where it prevents hypercalcemia by shutting down calcium mobilization from bone.
• In addition, CT serves as an important counter-regulatory hormone to PTH to prevent hypercalcemia and kidney calcification.
• CT is also found in the brain and spinal fluid where it may function as a neuromodulator. Intraventricular injection of CT inhibits feeding behavior in animals. CT could be a “calcium satiety hormone” analogous to the general satiety role proposed for CCK in brain. Consistent with this notion is the fact that the CT structure reveals a C-terminal amidation that is a signature for neuromodulator peptides; its N-terminal, intrachain disulfide is also characteristic of such molecules. This provides another example of a possible link between the endocrine function and behavioral response/CNS activity of peptide neuromodulators.

Question 17

CT Secretion

Answer 17

• CT is secreted by the thyroid C-cells under conditions of hypercalcemia.
• The thyroid C-cells contain a calcium sensor (receptor, CaR) that is identical to that in the parathyroid chief cells. In this instance sensing of calcium increases secretion of CT.
• When serum calcium is low, CT secretion is shut off.

Question 18

Biochemical Action of CT - Bone

Answer 18

• In bone, CT operates via receptors that are coupled to adenylyl cyclase through Gs and thereby raises the cAMP concentration.
• Although the receptors are distinct, and each is specific for its respective PTH or CT ligand, how can CT act on bone to lower calcium resorption by opposing PTH when both function via Gs to raise cAMP?

-The two opposing hormones can act via the same signal transduction pathway in the same tissue by affecting different cell types.

• CT raises cAMP in bone resorbing osteoclasts to directly inhibit their activity, while PTH increases cAMP in osteoblasts to cause them to secrete paracrine resorptive factors that in turn activate osteoclasts.
• Thus CT blocks PTH action distally at the osteoclast.

Question 19

CT Gene

Answer 19

• The CT gene also generates another neuropeptide, calcitonin gene related peptide (CGRP).
• CGRP is a neuromodulator and is the most potent and persistent vasodilator known.
• Either CGRP or CT can arise from the CT gene by alternative splicing of mRNAs.
• In thyroid “C” cells, the primary transcript is processed and spliced to yield an mRNA that is translated into prepro-calcitonin, while in certain neurons in the brain and spinal cord, the transcript is processed differently to exclude the CT coding exon and substitute the CGRP coding exon – with the result being prepro-CGRP.

Question 20

Vitamin D

Answer 20

•Though Vitamin D is considered a fat-soluble vitamin along with A, E and K, this classification is somewhat tenuous because, with adequate sunlight exposure, humans do not require a daily dietary supply. In this way Vitamin D is obtained as an “exo-prohormone” when UV light photolyzes 7-dehydrocholesterol in the epidermis (non-enzymatic) to vitamin D3.

Question 21

Dietary Vitamin D

Answer 21

• In individuals who have insufficient exposure to UV light including use of high SPF sunblockers, dietary sources of Vitamin D are critical to avoid deficiency.
• Vitamin D is found in fortified food products, oily fish [e.g., salmon], and eggs [though a minor source].
• Infant formulas must also contain sufficient vitamin D. In the US, milk fortified with vitamin D2 (ergocalciferol, a plant steroid) or vitamin D3 (cholecalciferol) is the principal source of dietary vitamin D.
• In other parts of the world, cereals and bread products may be fortified.
• Fat-soluble vitamins, such as vitamin D, are incorporated into micelles, absorbed by intestinal enterocytes and packaged into chylomicrons.
• The chylomicron remnant travels to the liver where it delivers the fat-soluble vitamins along with cholesterol.
• A variety of malabsorption disorders can impair uptake of digested materials and lead to a nutritional deficiency of vitamin D as well as other fat-soluble vitamins.

Question 22

Processing of Vitamin D to its Active Form

Answer 22

• Vitamin D itself does not act upon intestine, kidney and bone, but must first be bioactivated via a two-step metabolism in liver and kidney.
• The kidney activation of vitamin D is the key reaction.
• Initial modification occurs in the liver, where vitamin D3 is hydroxylated to the intermediate form, 25(OH)D3.
• This intermediate is then converted, by a kidney mitochondrial P450 1-hydroxylase enzyme (1-OHase) into one, 25(OH)2D3, the most potent form of vitamin D.
• When blood calcium and/or phosphate are elevated and the kidney 1-OHase is feedback repressed, the 25(OH)D3 intermediate is converted instead to the inactive metabolite 24, 25(OH)2D3.
• Formation of 24,25(OH)2D3 occurs in the kidney in a reaction catalyzed by 24- hydroxylase.

Question 23

Physiological Regulation of Vitamin D Production

• low dietary calcium?
• low dietary phosphate?
• active growth in children?
• pregnancy/lactation?

Answer 23

• Increased circulating 1,25(OH)2D3 raises calcium (and phosphate) to meet augmented needs of the minerals for the fetus in pregnancy, in milk for lactation, or to compensate for a relative dietary lack of the mineral.
• Estrogen inhibits bone resorption and directs 1,25(OH)2D3 action more to the gut, where calcium and phosphate are acquired from the diet.
• Physiologically, feedback loops of increased calcium (or phosphate) shut down production of 1,25(OH)2D3 or the hormone can limit its own biosynthesis at the parathyroids (decreased PTH gene transcription) or the kidney.
• When calcium (or phosphate) are high so that 1,25(OH)2D3 is elevated, 25(OH)D3 is metabolized to the inactive 24,25(OH)2D3 form

Question 24

Functions of Vitamin D

Answer 24

• Vitamin D, in addition to stimulating intestinal calcium absorption, renal calcium conservation and bone calcium resorption (hypercalcemic actions), stimulates intestinal absorption of phosphate and renal reabsorption of phosphate (hyperphosphatemic actions).
• In this way, vitamin D prevents rickets in children by raising the ion product Ca•PO4 in blood.
• Improving the Ca•PO4 matrix elicits proper bone mineral deposition through hydroxyapatite crystallization on the type I collagen organic matrix.
• When stressed by weight bearing, rachitic bones bend, but do not fracture in the traditional sense. Instead they incur numerous radiographically detectable lesions called pseudofractures.

Question 25

1,25(OH)2D3 is classified as the active hormonal form of vitamin D because it:

Answer 25

1. stimulates calcium absorption more rapidly than any other vitamin D form
2. is more active on a molar basis than any other form
3. production by kidney is regulated, according to the needs for calcium and/or phosphate
4. specific intracellular receptors in intestine, bone and kidney cells
5. chemically resembles steroid hormones [but lacks 4-ring structure]

Question 26

Biochemical Mode of 1,25(OH)2D3 Action in Intestinal Epithelial Cells

Answer 26

• Transport of calcium from lumen to blood occurs primarily in the proximal intestine.
• Initially, calcium moves passively from the lumen into the cell down its electrochemical gradient through epithelial Ca2+ channels.
• After entry, calcium attaches to the Calcium Binding Protein (CaBP).
• This sequestration keeps the free calcium concentration low within the cell.
• CaBP facilitates entry of Ca2+ into the intestinal cell and keeps Ca2+ from binding to negatively charged substances in the cell such as polar head groups of membrane lipids.
• Calcium is pumped to the blood primarily via a calcium ATPase and secondarily by Na+ -calcium exchange on the basolateral membrane.
• The capacity for intestinal calcium uptake primarily depends on the expression of both the amount of calcium ATPase and calcium binding proteins.
• Mg2+ interacts competitively with calcium transport.

-Consequently, a high concentration of Mg2+ in the lumen [some antacids; Mg(OH)2] can diminish net calcium uptake.

Question 27

Action of 1,25(OH)2D3

Answer 27

• Like all lipophilic hormones, the action of 1,25(OH)2D3 requires binding to intracellular receptors.
• The mechanism of action involves binding to a nuclear receptor with subsequent stimulation of DNA transcription.
• The vitamin D receptor (VDR) binds to DNA as a heterodimer with RXR that binds cis-retinoic acid.
• In intestinal epithelial cells, 1,25(OH)2D3 induces several proteins that contribute to the transcellular migration of calcium from the intestinal lumen to the blood.

Question 28

Proteins Induced in the Intestinal Lumen by 1,25(OH)2D3

Answer 28

•Epithelial calcium channel

-Located at the apical membrane (brush border) to facilitate Ca2+ entry from the lumen

•Calbindin -CaBP

-Cytoplasmic calcium binding protein that traps and ferries Ca2+ from the apical to the basolateral membrane

•Ca2+ - ATPase

-Located on the basolateral surface to pump Ca2+ out of the cell into the blood

Question 29

Biochemical Mode of 1,25(OH)2D3 Action in Bone Cells

Answer 29

• In bone, 1,25(OH)2D3 stimulates the osteoblastic synthesis and release of IL-6 and osteocalcin, after binding to its nuclear receptor.
• Osteocalcin is a bone-specific gamma carboxyglutamic acid (GLA)-containing protein that depends on vitamin K, analogous to blood clotting factors.
• IL-6 activates osteoclasts, and osteocalcin attaches to mineralized bone to recruit the osteoclasts via chemotaxis.
• Therefore, like PTH, excess 1,25(OH)2D3 causes the osteoblastic secretion of paracrine stimulators of osteoclastic bone resorption.

Question 30

Vitamin D Deficiency - Rickets

Answer 30

• Deficiency of 1,25(OH)2D3 can occur due to multiple factors and be congenital or acquired. In children, lack of sufficient dietary vitamin D is a major cause of rickets, which is simply a failure of mineralization of growing bone and cartilage.
• Severity determines the extent of symptoms that can include pain, irritability, delay in motor development, and poor growth.
• Bone problems in younger children include delayed closure of the fontanelles (i.e., the soft spot on the infant’s skull), frontal bossing (i.e., pronounced forehead), prominent costochondral margins, widening of ankles and wrists, bowlegs, and knock-knees.
• Radiologically, bones show low density, lost demarcation between metaphyses and growth plate, and loss of the provisional zone of calcification.

Question 31

Vitamin D Deficiency -Osteomalacia

Answer 31

• In adults, vitamin D deficiency is reportedly pandemic.
• Deficiency can be linked to factors other than diet and sun exposure such as sedentary lifestyle and stress.
• Low circulating vitamin D poses a risk factor not only for osteomalacia, but also for other problems including autoimmune, neurodegenerative, and cardiovascular diseases.

Question 32

Vitamin D Deficiency - Impaired Availability

Answer 32

• Lack of sufficient Vitamin D can result from congenital or acquired causes.
• Reduced availability of vitamin D can be caused by insufficient dietary intake, reduced exposure to sun light resulting in less de novo synthesis, or malabsorption.

-Fat malabsorption, in the absence of sufficient photoisomerization, as occurs in celiac disease, Crohn disease, pancreatic insufficiency, cholestatic liver disease, abetalipoproteinemia, cystic fibrosis, and lymphatic pathology leads to a deficiency of circulating vitamin D3 as a precursor to formation of 25(OH)D3.

•The prevalence of vitamin D deficiency among racial groups correlates with differences in skin pigmentation.

• The highest prevalence of vitamin D deficiency rickets has been reported in Black children, followed by Asian Indian and then white.
• Increased melanin with darker skin pigmentation acts as a natural sun blocker. Latitude of residence and the season (i.e., winter vs summer) affects photoisomerization, as well.
• Sunblockers with SPF of >30 can reduce this synthetic capacity by as much as 95%.
• Since vitamin D is transferred across the placenta to the fetus, reduced vitamin D stores in the mother can affect bone growth in the fetus.
• This maternal deficiency can be a major problem in dark-skinned pregnant women at higher latitudes in winter months as three factors come in to play reducing de novo synthesis.
• Vitamin D concentrations tend to be low in premature infants due to less time to have obtained maternal vitamin D.
• Breast milk has a low content of vitamin D even in mothers who have sufficient circulating vitamin D.

Question 33

Hypervitaminosis D

Answer 33

• Consuming very high doses of vitamin D can lead to hypervitaminosis D.
• Because vitamin D is a fat-soluble vitamin, when in excess it cannot easily be excreted and instead accumulates in fat tissue.
• The resulting abnormally high circulating calcium resulting from increased intestinal absorption, renal reabsorption and especially bone resorption in this condition can lead to severe bone damage and damage to soft tissues and kidney.
• Symptoms of hypervitaminosis D include constipation, decreased appetite, dehydration, fatigue, irritability, and muscle weakness. Tests show elevated serum concentration of 1,25(OH)2D3, calcium and phosphorus.